The mycobacterial cell wall is profoundly altered in response to environmental conditions; however, cell wall regulatory mechanisms, which cause antibiotic tolerance, are not well understood. Our long-term goal is to build a comprehensive molecular model of mycobacterial cell wall regulation in clinically relevant stresses. The objective of this proposal is to determine how two essential factors, FtsQ and SepIVA, contribute to the regulation of cell elongation and division during stress in Mycobacterium smegmatis. The central hypothesis of this proposal is that FtsQ and SepIVA are cell division regulators, are post-translationally modified in response to stress, and that these modifications can lead to altered cell division behavior and stress tolerance. This hypothesis was formulated based on preliminary data showing that FtsQ phosphorylation impairs survival under antibiotic stress, and that the arginine methylations of SepIVA promote survival in stationary phase. The rationale behind this research is that knowledge of the signaling events that allow mycobacteria to respond to stress will lead to a better understanding of mycobacterial infection physiology and mechanisms of antibiotic tolerance, which could lead to the development of new anti-mycobacterial drugs. The research in this proposal pursues two specific aims: Aim 1) Determine how phosphorylation of FtsQ contributes to the regulation of cell division and cell wall metabolism. The data suggest that phosphorylation of FtsQ alters the activity of cell wall enzymes under stress, leading to changes in cell division and stress tolerance. This work will: a) assess how FtsQ phosphorylation impacts cell division and survival in stresses and antibiotic treatment, b) identify the kinase of FtsQ and the conditions under which it is phosphorylated, and c) characterize how FtsQ phosphorylation affects protein interactions and divisome assembly. Aim 2) Characterize the mechanism of SepIVA?s function in cell division, and determine how arginine methylation affects this function. The data suggest that SepIVA regulates cell wall precursor enzymes required for cell division, that this regulation contributes to survival under stress, and that arginine methylations on SepIVA help modulate its function. This work will: a) Identify the stage of cell wall synthesis that SepIVA regulates and determine how SepIVA affects the localization of other cell division factors, b) assess how the post-translational regulation of SepIVA alters its localization, survival under stress, and role in cell division, and c) identify SepIVA interaction partners and determine how arginine methylation affects protein interactions. This work takes an innovative approach to the study of cell division regulation by focusing on post-translational modifications of core septal factors - including arginine methylations, which have never before been described in bacteria. The results of this work will illuminate cell wall regulatory mechanisms that are likely to be important for infection and antibiotic tolerance in pathogenic mycobacteria.